Elise G. Lavoie

Comparative hydrolysis of P2 receptor agonists by NTPDases 1, 2, 3 and 8

Nucleoside triphosphate diphosphohydrolases 1, 2, 3 and 8 (NTPDases 1, 2, 3 and 8) are the dominant ectonucleotidases and thereby expected to play important roles in nucleotide signaling. Distinct biochemical characteristics of individual NTPDases should allow them to regulate P2 receptor activation differentially. Therefore, the biochemical and kinetic properties of these enzymes were compared. NTPDases 1, 2, 3 and 8 efficiently hydrolyzed ATP and UTP with Km values in the micromolar range, indicating that they should terminate the effects exerted by these nucleotide agonists at P2X1- and P2Y2,4,11 receptors. Since NTPDase1 does not allow accumulation of ADP, it should terminate the activation of P2Y1,12,13 receptors far more efficiently than the other NTPDases. In contrast, NTPDases 2, 3 and 8 are expected to promote the activation of ADP specific receptors, because in the presence of ATP they produce a sustained (NTPDase2) or transient (NTPDases 3 and 8) accumulation of ADP. Interestingly, all plasma membrane NTPDases dephosphorylate UTP with a significant accumulation of UDP, favoring P2Y6 receptor activation. NTPDases differ in divalent cation and pH dependence, although all are active in the pH range of 7.0-.5. Various NTPDases may also distinctly affect formation of extracellular adenosine and therefore adenosine receptor-mediated responses, since they generate different amounts of the substrate (AMP) and inhibitor (ADP) of ecto-5-nucleotidase, the rate limiting enzyme in the production of adenosine. Taken together, these data indicate that plasma membrane NTPDases hydrolyze nucleotides in a distinctive manner and may therefore differentially regulate P2 and adenosine receptor signaling.

Nucleoside triphosphate diphosphohydrolase-2 is the ecto-ATPase of type I cells in taste buds.

The presence of one or more calcium‐dependent ecto‐ATPases (enzymes that hydrolyze extracellular 5′‐triphosphates) in mammalian taste buds was first shown histochemically. Recent studies have established that dominant ecto‐ATPases consist of enzymes now called nucleoside triphosphate diphosphohydrolases (NTPDases). Massively parallel signature sequencing (MPSS) from murine taste epithelium provided molecular evidence suggesting that NTPDase2 is the most likely member present in mouse taste papillae. Immunocytochemical and enzyme histochemical staining verified the presence of NTPDase2 associated with plasma membranes in a large number of cells within all mouse taste buds. To determine which of the three taste cell types expresses this enzyme, double‐label assays were performed with antisera directed against the glial glutamate/aspartate transporter (GLAST), the transduction pathway proteins phospholipase Cβ2 (PLCβ2) or the G‐protein subunit α‐gustducin, and serotonin (5HT) as markers of type I, II, and III taste cells, respectively. Analysis of the double‐labeled sections indicates that NTPDase2 immunoreactivity is found on cell processes that often envelop other taste cells, reminiscent of type I cells. In agreement with this observation, NTPDase2 was located to the same membrane as GLAST, indicating that this enzyme is present in type I cells. The presence of ecto‐ATPase in taste buds likely reflects the importance of ATP as an intercellular signaling molecule in this system. J. Comp. Neurol. 497:1–12, 2006.

Specificity of the ecto-ATPase inhibitor ARL 67156 on human and mouse ectonucleotidases

ARL 67156, 6‐N,N‐Diethyl‐D‐β‐γ‐dibromomethylene adenosine triphosphate, originally named FPL 67156, is the only commercially available inhibitor of ecto‐ATPases. Since the first report on this molecule, various ectonucleotidases responsible for the hydrolysis of ATP at the cell surface have been cloned and characterized. In this work, we identified the ectonucleotidases inhibited by ARL 67156.

Innate immune responses to Pseudomonas aeruginosa infection.

Innate immune responses play a critical role in controlling acute infections due to Pseudomonas aeruginosa in both mice and in humans. In this review we focus on innate immune recognition and clearance mechanisms that are important for controlling P. aeruginosa in the mammalian lung, with particular attention to those that influence the outcome of inxa0vivo infection in murine models.

Portal fibroblasts regulate the proliferation of bile duct epithelia via expression of NTPDase2

Bile duct epithelia are the target of a number of “cholangiopathies” characterized by disordered bile ductular proliferation. Although mechanisms for bile ductular proliferation are unknown, recent evidence suggests that extracellular nucleotides regulate cell proliferation via activation of P2Y receptors. Portal fibroblasts may regulate bile duct epithelial P2Y receptors via expression of the ecto-nucleotidase NTPDase2. Thus, we tested the hypothesis that portal fibroblasts regulate bile duct epithelial proliferation via expression of NTPDase2. We generated a novel co-culture model of Mz-ChA-1 human cholangiocarcinoma cells and primary portal fibroblasts. Cell proliferation was measured by bromodeoxyuridine uptake. NTPDase2 expression was assessed by immunofluorescence and quantitative real-time reverse transcription PCR. NTPDase2 expression in portal fibroblasts was blocked using short interfering RNA. NTPDase2 overexpression in portal myofibroblasts isolated from bile duct-ligated rats was achieved by cDNA transfection. Co-culture of Mz-ChA-1 cells with portal fibroblasts decreased their proliferation to 26% of control. Similar decreases in Mz-ChA-1 proliferation were induced by the soluble ecto-nucleotidase apyrase and the P2 receptor inhibitor suramin. The proliferation of Mz-ChA-1 cells returned to baseline when NTPDase2 expression in portal fibroblasts was inhibited using NTPDase2-specific short interfering RNA. Untransfected portal myofibroblasts lacking NTPDase2 had no effect on Mz-ChA-1 proliferation, yet portal myofibroblasts transfected with NTPDase2 cDNA inhibited Mz-ChA-1 proliferation. We conclude that portal fibroblasts inhibit bile ductular proliferation via expression of NTPDase2 and blockade of P2Y activation. Loss of NTPDase2 may mediate the bile ductular proliferation typical of obstructive cholestasis. This novel cross-talk signaling pathway may mediate pathologic alterations in bile ductular proliferation in other cholangiopathic conditions.

P2Y2 Receptor Transcription Is Increased by NF-κB and Stimulates Cyclooxygenase-2 Expression and PGE2 Released by Intestinal Epithelial Cells

Inflammatory stresses associated with inflammatory bowel diseases up-regulate P2Y2 mRNA receptor expression in the human colon adenocarcinoma cell line Caco-2, the noncancerous IEC-6 cells and in colonic tissues of patient suffering from Crohn’s disease and ulcerative colitis. However, the transcriptional events regulating P2Y2 receptor (P2Y2R) expression are not known. We have identified a putative transcription start site in the P2Y2R gene and demonstrated acetylation of Lys14 on histone H3 and Lys8 on histone H4, thus suggesting that the chromatin associated with the P2Y2 promoter is accessible to transcription factors. We also showed that the transcription factor NF-κB p65 regulates P2Y2R transcription under both proinflammatory and basal conditions. A NF-κB-responsive element was identified at −181 to −172 bp in the promoter region of P2Y2. Hence, activation of P2Y2R by ATP and UTP stimulated cyclooxygenase-2 expression and PGE2 secretion by intestinal epithelial cells. These findings demonstrate that P2Y2R expression is regulated during intestinal inflammation through an NF-κB p65-dependent mechanism and could contribute not only to inflammatory bowel disease but also to other inflammatory diseases by regulating PG release.

Ectonucleotidases in the digestive system: Focus on NTPDase3 localization

Extracellular nucleotides and adenosine are biologically active molecules that bind members of the P2 and P1 receptor families, respectively. In the digestive system, these receptors modulate various functions, including salivary, gastric, and intestinal epithelial secretion and enteric neurotransmission. The availability of P1 and P2 ligands is modulated by ectonucleotidases, enzymes that hydrolyze extracellular nucleotides into nucleosides. Nucleoside triphosphate diphosphohydrolases (NTPDases) and ecto-5-nucleotidase are the dominant ectonucleotidases at physiological pH. While there is some information about the localization of ecto-5-nucleotidase and NTPDase1 and -2, the distribution of NTPDase3 in the digestive system is unknown. We examined the localization of these ectonucleotidases, with a focus on NTPDase3, in the gastrointestinal tract and salivary glands. NTPDase1, -2, and -3 are responsible for ecto-ATPase activity in these tissues. Semiquantitative RT-PCR, immunohistochemistry, and in situ enzyme activity revealed the presence of NTPDase3 in some epithelial cells in serous acini of salivary glands and mucous acini and duct cells of sublingual salivary glands, in cells from the stratified esophageal and forestomach epithelia, and in some enteroendocrine cells of the gastric antrum. Interestingly, NTPDase2 and ecto-5-nucleotidase are coexpressed with NTPDase3 in salivary gland cells and stratified epithelia. In the colon, neurons express NTPDase3 and glial cells express NTPDase2. Ca(2+) imaging experiments demonstrate that NTPDases regulate P2 receptor ligand availability in the enteric nervous system. In summary, the specific localization of NTPDase3 in the digestive system suggests functional roles of the enzyme, in association with NTPDase2 and ecto-5-nucleotidase, in epithelial functions such as secretion and in enteric neurotransmission.

Localization of plasma membrane bound NTPDases in the murine reproductive tract.

Extracellular nucleotides might influence aspects of the biology of reproduction in that ATP affects smooth muscle contraction, participates in steroidogenesis and spermatogenesis, and also regulates transepithelial transport, as in oviducts. Activation of cellular nucleotide purinergic receptors is influenced by four plasma membrane-bound members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family, namely NTPDase1, NTPDase2, NTPDase3, and NTPDase8 that differ in their ecto-enzymatic properties. The purpose of this study was to characterize the expression profile of the membrane-bound NTPDases in the murine female and male reproductive tracts by immunological techniques (immunolabelling, Western blotting) and by enzymatic assays, in situ and on tissue homogenates. Other than the expected expression on vascular endothelial and smooth muscle cells, NTPDase1 was also detected in Sertoli cells and interstitial macrophages in testes, in ovarian granulosa cells, and in apical cells from epididymal epithelium. NTPDase2 was largely expressed by cells in the connective tissue; NTPDase3 in secretory epithelia, and finally, NTPDase8 was not detected in any of the tissues studied here. In addition, NTPDase6 was putatively detected in Golgi-phase acrosome vesicles of round spermatids. This descriptive study suggests close regulation of extracellular nucleotide levels in the genital tract by NTPDases that may impact specific biological functions.

Contribution of Myofibroblasts of Different Origins to Liver Fibrosis

The most common cause of liver failure is cirrhosis, due to progressive liver fibrosis and other architectural changes in the liver. Fibrosis occurs after liver injury or stress and results directly from an imbalance between the processes of extracellular matrix synthesis (fibrogenesis) and degradation (fibrolysis). Although research studies have identified several promising targets at the molecular level, current therapies to prevent and treat hepatic fibrosis in patients have only shown limited success. It is well established that liver myofibroblasts (MFs) are the primary effector cells responsible for the extensive extracellular matrix accumulation and scar formation observed during hepatic fibrosis, in both clinical and experimental settings. Thus, as the major fibrogenic cells implicated in wound healing and tissue repair response, liver MFs could represent excellent targets for antifibrotic therapies. Still, the exact nature and identities of liver MFs precursors have yet to be resolved, and their relative contribution to hepatic fibrosis to be determined. The goal of this review is to examine the relative importance of liver MF precursors in the pathogenesis of liver fibrosis.

Immunocytochemical localization of NTPDases1 and 2 in the neural retina of mouse and zebrafish

Ectonucleoside triphosphate diphosphohydrolases (E‐NTPDases) are a family of membrane‐bound enzymes that hydrolyze extracellular di‐ and triphosphate nucleosides. E‐NTPDases have been proposed to control extracellular nucleotide levels that mediate intercellular communication by binding to specific membrane receptors. Here we show a detailed immunocytochemical localization of two enzymes of the E‐NTPDase family in the retinal layers of two vertebrate species, namely, the mouse and the zebrafish. In the mouse retina, NTPDase2 was chiefly localized in Müller glia and ganglion cell processes. NTPDase1 was located on neurons as well, since it was expressed by horizontal and ganglion cell processes, suggesting that nucleotides such as ATP and ADP can be hydrolyzed at the surface of these cells. NTPDase1 was also detected in intraretinal blood vessels of the mouse. Regarding zebrafish, NTPDases1 and 2 seem to be differentially localized in horizontal cell processes, photoreceptor segments, and ganglion cell dendrites and axons, but absent from Müller glia. Moreover, NTPDases1 and 2 appear to be expressed within the germinal margin of the zebrafish retina that contains proliferative and differentiating cells. Retinal homogenates from both species exhibited ecto‐ATPase activity which might be attributed at least to NTPDases1 and 2, whose expression is described in this report. Our results suggest a compartmentalized regulation of extracellular nucleotide/nucleoside concentration in the retinal layers, supporting a relevant role for extracellular nucleotide mediated‐signaling in vertebrate retinas. Synapse 63:291–307, 2009.